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J Biol Chem, Vol. 273, Issue 43, 28019-28024, October 23, 1998
From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
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ABSTRACT |
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Abstract
Introduction
Procedures
Results
Discussion
References
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In the budding yeast Saccharomyces
cerevisiae, association with the 70-kDa cyclase-associated
protein (CAP) is required for proper response of adenylyl cyclase to
Ras proteins. We show here that a small segment comprising the
N-terminal 36 amino acid residues of CAP is sufficient for association
with adenylyl cyclase as well as for its function in the Ras-adenylyl
cyclase pathway as assayed by the ability to confer
RAS2Val-19-dependent heat shock
sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase
was mapped to a segment of 119 amino acid residues near its C terminus.
Both of these regions contained tandem repetitions of a heptad motif
XXXXX (where represents a hydrophobic
amino acid and X represents any amino acid), suggesting a
coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly
mutagenized CAP, they were found to carry substitution mutations in one
of the key hydrophobic residues in the heptad repeats. Furthermore,
mutations of the key hydrophobic residues in the heptad repeats of
adenylyl cyclase also resulted in loss of association with CAP. These
results indicate the coiled-coil mechanism as a basis of the
CAP-adenylyl cyclase interaction.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The budding yeast Saccharomyces cerevisiae has two RAS genes, RAS1 and RAS2, whose protein products are structurally, functionally, and biochemically similar to mammalian Ras proto-oncoproteins (for reviews, see Refs. 1 and 2). The yeast Ras proteins are essential regulatory elements of adenylyl cyclase, which catalyzes the production of cAMP, a second messenger vital for cell growth (3, 4). The Ras-adenylyl cyclase pathway has been implicated in transduction of a signal triggered by glucose to an intracellular environment where a protein phosphorylation cascade is induced by cAMP. Yeast cells bearing the activated RAS2 gene, RAS2Val-19, exhibit an elevated level of intracellular cAMP and display abnormal phenotypes, including sensitivity to heat shock, sensitivity to nutritional starvation, and failure to sporulate (3, 5).
Yeast adenylyl cyclase, encoded by the CYR1 gene, consists
of 2026-amino acid residues that comprise at least four domains: the
N-terminal, the middle leucine-rich repeat, the catalytic, and the
C-terminal domains (6, 7). The leucine-rich repeat domain contains a
binding site for Ras proteins (8, 9). Adenylyl cyclase forms a complex
with 70-kDa CAP.1 CAP was
identified biochemically as the only protein associated tightly with
adenylyl cyclase and also by genetic screening of a gene whose mutation
abolished the RAS2Val-19-dependent
heat shock sensitivity (10, 11). Studies on the function of CAP
revealed that CAP is a multifunctional protein. It was shown that the
N-terminal region, mapped to residues 1-168, is required for
acquisition of heat shock sensitivity in the
RAS2Val-19 background while the C-terminal
region, mapped to residues 369-526, is required for normal cell
morphology and responsiveness to nutrient deprivation and excess (12).
The C-terminal function appears to be related to regulation of the
actin cytoskeleton as evidenced by complementation of its defect by
overexpression of profilin or SNC1 (13, 14) and by demonstration of its
direct association with actin monomer and of its actin-sequestering
activity (15, 16). In addition, CAP possesses two proline-rich
sequences in its middle region intervening between the two regions,
with which associations of actin-binding protein 1, elongation factor
1, and ribosomal protein L3 were recently shown (17, 18).
The N-terminal region of CAP binds to the C-terminal region of adenylyl cyclase (19), and this association appears to be required for the proper in vivo response of adenylyl cyclase to Ras, because its loss by mutation of either CAP or adenylyl cyclase resulted in disappearance of the RAS2Val-19-dependent heat shock sensitivity and in a reduced cAMP response to glucose stimulation (19). This function resides solely in the CAP N-terminal region and is separable from the functions of the other regions as reported (12). We have recently shown biochemically that the association of adenylyl cyclase with the CAP N-terminal region is responsible for efficient stimulation of adenylyl cyclase activity by the posttranslationally modified form of Ras, although the molecular mechanism underlying this process remains to be clarified (20). In this report, we have mapped a minimal region of CAP responsible for its N-terminal function and analyzed the molecular mechanism for its association with adenylyl cyclase.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Cell Strains and Growth Media--
The S. cerevisiae
strains used are listed in Table I.
Replacement of the chromosomal CAP gene with its N-terminal
deletion mutant CAP
N-1 was carried out as described
previously (20). The resulting yeast strain expresses only the
C-terminal segment of CAP corresponding to residues 369-526 under
control of the yeast ADC1 promoter. Yeast cells were grown
in YPD (2% Bacto-peptone, 1% Bacto-yeast extract, 2% glucose) or
yeast synthetic medium (0.67% yeast nitrogen base, 2% glucose) with
appropriate auxotrophic supplements. Genetic manipulations of yeast
cells were performed as described previously (21). Transformation into
yeast cells was carried out with lithium acetate (22).
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Construction of Expression Plasmids and Oligonucleotide-directed
Mutagenesis--
A plasmid, pAD-GST-CAP (18), was used to express the
full-length CAP in yeast as a fusion protein with GST under control of
the ADC1 promoter. Various deletions were introduced into
the CAP gene by cleavage of pAD-GST-CAP with suitable pairs
of restriction endonucleases and resealing by T4 DNA ligase with a
linker oligonucleotide, 5'-CTAGTCTAGACTAG-3', bearing stop codons in
all reading frames, between. The resulting plasmids were designated as
pAD-GST-CAP-(x-x)s, where x-x represents the
range of the expressed CAP polypeptides in amino acid positions. A
5'-terminal 107-base pair fragment corresponding to residues 1-36 of
CAP, CAP-(1-36), was amplified by PCR (23) using suitable
oligonucleotide primers and, after cleavage with BamHI and
SmaI in the primer sequences, cloned into pAD-GST to produce
pAD-GST-CAP-(1-36). Similarly, DNA fragments encoding various
C-terminal polypeptides of adenylyl cyclase were amplified by PCR using
suitable primers and cloned into pAD-GST to produce
pAD-GST-CYR1-(y-y), where y-y represented the
range of the expressed adenylyl cyclase polypeptide in amino acid
positions. Specific amino acid substitution mutations were introduced
into adenylyl cyclase by the gapped duplex method using suitable
mutagenic oligonucleotides (24). The mutant genes were used to replace the corresponding wild-type genes in the expression plasmids. YEP24-ADC1-CYR1-(1-40, 1769-2026) and YEP24-ADC1-CYR1-(1-40,
606-2026), which expressed adenylyl cyclase carrying internal
deletions of residues 41-1768 and 41-605, were identical to
YEP24-ADC1-CYR1-(41-1768) and YEP24-ADC1-CYR1-(41-605),
respectively, described previously (8, 19).
Yeast Two-hybrid Assay--
The various mutant CAP
genes were transferred to pGBT9 or pGBT10 vector (25) for expression of
the corresponding polypeptides as GBT fusions in yeast. Similarly, the
various mutant CYR1 genes were transferred to pGAD-GH (25)
for expression as GAD fusions. The resulting plasmids were designated
as pGBT-CAP-(x-x) or pGAD-CYR1-(y-y), respectively. The reporter yeast strain YPB2(CAPN) was cotransformed with pGAD-CYR1-(y-y) and pGBT-CAP-(x-x), and
the resulting Trp+, Leu+-transformants were
assayed for 
-galactosidase activity by a filter assay as described
(25).
Random Mutagenesis of CAP-- A DNA fragment corresponding to CAP-(1-77), was subjected to an error-prone PCR to introduce random mutations as described before (26). The amplified fragments were cleaved with BamHI and SmaI present in the PCR primers, cloned into matching cleavage sites of pGBT10, and examined for interaction with pGAD-CYR1-(1879-2026) by the yeast two-hybrid assay as described above. The CAP mutants that failed to interact with CYR1(1879-2026) were characterized by DNA sequencing and transferred to pAD-GST for expression as GST-fusion proteins in yeast cells.
Measurement of in Vivo Association between CAP and Adenylyl Cyclase-- Yeast FS1 was transformed with a combination of either YEP24-ADC1-CYR1-(1-40, 1769-2026) or YEP24-ADC1-CYR1-(1-40, 606-2026) and one of the pAD-GST-CAP-(x-x) plasmids bearing various mutations. In another series of experiments, SP1 was transformed with pAD-GST-CYR1-(y-y) bearing various mutations. The resulting transformants were grown to a density of 1 × 107 cells/ml, harvested by centrifugation, and disrupted by shaking with glass beads in buffer C (50 mM MES, pH 6.2, 0.1 mM MgCl2, 0.1 mM EGTA, 2 mM dithiothreitol, 10% glycerol) as described previously (9, 19). Crude membrane fraction was prepared by centrifugation of the homogenate at 27,000 × g for 80 min. GST-CAP-(x-x) or GST-CYR1-(y-y) protein was solubilized from the crude membrane fraction with buffer C containing 1% Lubrol PX, 0.5 M NaCl, and 1 mM phenylmethylsulfonyl fluoride, adsorbed onto glutathione-Sepharose resin, and eluted with 20 mM glutathione as described (27). Proteins bound to the GST-fusion proteins, which were co-eluted from the resin, were subjected to Western immunoblot detection of CYR1 or CAP by using specific antibodies: rabbit polyclonal antisera for a 15-amino acid synthetic peptide of the C terminus of adenylyl cyclase (anti-CYR1CT) or for the full-length CAP (anti-CAP), respectively, as described previously (19). A rabbit polyclonal antiserum for GST (anti-GST) was used for detection of GST fusion proteins.
Other Methods-- Survival of yeast cells after heat shock treatment at 55 °C for 5 min was examined by a replica plating method as described previously (3, 19). SDS-polyacrylamide gel electrophoresis and Western immunoblot analysis were performed as described (28, 29). The ECL immunodetection system (Amersham Pharmacia Biotech) was used for signal development.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Mapping of the Mutual Binding Sites of CAP and Adenylyl
Cyclase--
Previous experiments had already mapped the N-terminal
function of CAP to residues 1-168 (12) and the CAP-binding site of adenylyl cyclase to residues 1879-2026 (19). To further delineate the
binding sites, we introduced various deletion mutations into CAP and
adenylyl cyclase as described under "Experimental Procedures." Interactions of the various CAP mutants with CYR1-(1879-2026) and of
the various CYR1 C-terminal mutants with CAP were examined by employing
the yeast two-hybrid system (Fig.
1A). As an indicator strain,
we used YPB2(CAPN), whose chromosomal CAP gene was
replaced by its N-terminal deletion mutant CAPN-1 in
order to exclude the possibility that endogenous CAP complexed with an
otherwise negative GAD-fusion CAP mutant may serve as a bridge to yield a positive interaction with the GBT-fusion CYR1. Formation of such a
CAP dimer had been reported before (16). To our surprise, the shortest
CAP construct carrying only the N-terminal 36 residues, CAP-(1-36), as
well as the longer CAP-(1-66), CAP-(1-77), and CAP-(1-88) exhibited
a positive interaction with the C-terminal region of adenylyl cyclase
(Fig. 1A). In contrast, CAP-(78-526) lacking the N-terminal
region did not exhibit any interaction. On the other hand, the shortest
fragment of CYR1 giving a positive interaction with CAP was 119 residues corresponding to positions 1898-2016 (Fig. 1A). An
N-terminal deletion up to position 1935 destroyed the activity to
interact with CAP.
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CAP-(1-36) Is Sufficient for Its N-terminal Function in
Vivo--
We examined the abilities of the CAP deletion mutants to
confer heat shock sensitivity to TK161-R2V(CAPN) cells, which
carried the CAPN-1 gene encoding the protein lacking its
N-terminal function and thereby were made resistant to heat shock in
the RAS2Val-19 background. As shown in Fig.
2A, expression of the shortest
fragment, GST-CAP-(1-36), as well as other longer CAP N-terminal
fragments was sufficient to restore the heat shock sensitivity in this
yeast strain. As observed in the binding assays, both GST-CAP-(77-526) and GST only were found inactive. This result implied that the N-terminal 36 residues are functional in the Ras-adenylyl cyclase pathway.
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Identification of Mutations That Abrogate the CAP-Adenylyl Cyclase
Interaction--
The mutually interacting regions, CAP-(1-36) and
CYR1-(1898-2016), were searched for a peculiar sequence motif hinting
at the mechanism of their interaction. The search identified tandem repetitions of a heptad motif XXXXX (where
and X represent a hydrophobic amino acid and any amino
acid, respectively; for reviews, see Refs. 30 and 31) in both residues
13-30 of CAP and residues 1916-1930 of adenylyl cyclase (Fig.
3A). If the heptad repeat
motif is taken as that of a leucine zipper LXXXXXX (32), adenylyl cyclase has one more repeat unit in residues 1931-1937. These
heptad repeats enabled us to predict formation of -helices that are
wound around each other to form a superhelix, the coiled-coil structure
(30, 31). This was also supported by calculation of the probability of
adopting a coiled-coil conformation using the computer program COILS
(33). Residues 11-33 of CAP were predicted to have more than 95%
probability of forming a coiled-coil, whereas the probability for the
other portion of CAP was almost 0.
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N) yeast cells (Fig.
5A). Strikingly, four out of
the five clones turned out to carry an amino acid substitution mutation
at Leu-20, Leu-27, or Val-30, all of which corresponded to the key
hydrophobic residues in the heptad repeats (Fig. 3A).
Moreover, the mutations were introduced in such a way that the
hydrophobic residues were converted to neutral or hydrophilic residues
(Table II). The other clone HS205 carried two mutations, T31P and Q34L.
Although Gln-34 is located at the position in the heptad repeats
corresponding to the key hydrophobic residue, it is presently unclear
which of the two mutations is responsible for the effect.
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-position (Fig. 3A) were converted to Ser and to either Pro or Arg, respectively, by oligonucleotide-directed mutagenesis. The
CYR1 C-terminal fragments carrying L1916S, L1923P, and L1923R mutations
all lost the ability to associate with CAP as assayed by the yeast
two-hybrid method (Fig. 4B) or by the in vivo
binding assay (Fig. 4C). Overexpression of CYR1-(1822-2026)
bearing the same mutations could not suppress the
RAS2Val-19-dependent heat shock
sensitivity (Fig. 5B). These results indicated that the
hydrophobic residues of both CAP and adenylyl cyclase are indeed
critical not only for their mutual association but also for their
proper function in the Ras-adenylyl cyclase pathway and further
supported the involvement of the coiled-coil mechanism for their
interaction.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We have shown that the N-terminal 36 residues of CAP are
sufficient for association with adenylyl cyclase as well as for its in vivo function in the Ras-adenylyl cyclase pathway. The
CAP-binding site of adenylyl cyclase was mapped to a 119-residue
segment near the C terminus. Close inspection of the primary sequences
of the two mutual binding sites has identified typical heptad repeat motifs (XXXXX)n indicative of a
coiled-coil interaction (30, 31) (Fig. 3A). Furthermore, the
presence of the coiled-coil in the CAP N terminus was predicted by the
computer program COILS (33). Coiled-coils are composed of two, three,
or four -helices wound around each other to form a left-handed
superhelix. This structure is found in a wide variety of proteins
including cytoskeletal structural proteins, transcription factors, etc.
(30, 31). Further, a number of proteins whose three-dimensional
structures have been determined are found to contain coiled-coil
segments, many of them very short. The presence of this structure in a
protein can be predicted from an array of the typical heptad motif in its amino acid sequence, which is labeled a-b-c-d-e-f-g,
where a and d are primarily hydrophobic, most
frequently Leu, residues and form the helix interface, while
b, c, e, f, and
g are hydrophilic and form the solvent-exposed surface of
the coiled-coil. The association of the helices is stabilized by
hydrophobic interactions at the side chains of the hydrophobic residues
a and d, which form an apolar stripe along one
side of the helix, and additionally by ionic interactions at the side
chains of the nearby charged residues. This is apparent in spinning
wheel representations of the two possibly interacting segments
CAP-(6-34) and CYR1-(1916-1940) (Fig. 3B). The assignments
of the a- and d-positions were supported by an
amino acid sequence homology between CAP-(6-34) and Dirofilaria immitis paramyosin (34), which was found by BLASTP search (35) of
GenBankTM entries. Residues 16-29 of CAP (Fig.
3A) shared nine identical residues with residues 213-226 of
paramyosin (LAQQLEEARRRLED). This match made it possible to predict
positions a-g of each residue of CAP from the proposed
-helical structure of paramyosin (36).
A further proof for the coiled-coil interaction came from the studies on mutations of CAP and adenylyl cyclase, which abrogated the interaction. Strikingly, the three residues of CAP (Leu-20, Leu-27, and Val-30) and two residues of adenylyl cyclase (Leu-1916 and Leu-1923) that were identified to be essential for the interaction based on these mutational studies are all hydrophobic and located at position a or d. These results strongly support the notion that the coiled-coil mechanism forms a molecular basis for the CAP-adenylyl cyclase interaction.
At present, it is impossible for us to predict from the amino acid sequences how many strands of CAP and adenylyl cyclase contribute to the formation of the coiled-coil superhelix. It is also impossible to predict the relative orientation, parallel or anti-parallel, of the strands of CAP and adenylyl cyclase and how individual pairs of the residues from each strand are formed, both of which are primarily determined by polar and ionic interactions between residues flanking the hydrophobic core (30, 31). Elucidation of these structural features awaits determination of the three-dimensional structure of the CAP-adenylyl cyclase complex.
The heptad repeat structure is well conserved in CAP homologues identified in other organisms including Schizosaccharomyces pombe and mammals, although the N-terminal function of the budding yeast CAP is not conserved among them (37-40). This suggests that in those organisms CAP may establish a coiled-coil interaction at its N-terminal short segment with a certain protein to exert a function that is presumably different from that of the Ras-adenylyl cyclase pathway. The identification of such a CAP-interacting protein may reveal a novel function of CAP in addition to its C-terminal cytoskeletal function, which is known to be conserved between yeasts and mammals (37-40).
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ACKNOWLEDGEMENTS |
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We thank X.-H. Deng for skillful technical assistance and A. Seki and A. Kawabe for help in preparation of this manuscript.
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FOOTNOTES |
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* This investigation was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Yamanouchi Foundation for Research on Metabolic Disease.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-78-341-7451 (ext. 3230); Fax: 81-78-341-3837; E-mail: kataoka{at}kobe-u.ac.jp.
The abbreviations used are: CAP, adenylyl cyclase-associated protein; CYR1, adenylyl cyclase; GST, glutathione S-transferasePCR, polymerase chain reactionGBT, GAL4 DNA-binding domainGAD, GAL4 transactivation domainMES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
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Abstract
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Procedures
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Discussion
References
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